Cardiotonic steroids (CTS) consist of a group of chemicals that specifically bind to the Na+/K+-ATPase. They include plant-derived digitalis drugs such as digoxin and ouabain and vertebrate-derived aglycone such as bufalin and marinobufagenin. Recent studies have identified both ouabain and marinobufagenin as endogenous steroids whose production and secretion are regulated by multiple physiological and pathological stimuli including angiotensin II and epinephrine in humans. These steroids can activate protein kinases and regulate cell growth, gene expression, intracellular calcium, and reactive oxygen species (ROS) concentrations, thus playing important roles in the control of renal and cardiovascular functions, protection of ischemia/reperfusion injury and stimulation or inhibition of cell growth.
Src family kinases are 52-62-kDa membrane-associated nonreceptor tyrosine kinases and they participate in several tyrosine phosphorylation-related signaling pathways in response to various extracellular ligands. Src, for example, contains at least three important protein interaction domains. The SH3 domain binds to polyproline motifs and the SH2 domain interacts with the phosphorylated tyrosine residues. The kinase domain reacts with the nucleotide and phosphorylates the substrate. Binding of protein ligands to the SH3 or SH2 domain can activate Src. Proteins that bind with kinase domain of Src were also reported to be capable of regulating Src activity.
Na+/K+-ATPase, the molecular machinery of the cellular sodium pump, belongs to a family of evolutionarily ancient enzymes that couple the hydrolysis of ATP to membrane ion translocation. It is now believed that the Na+/K+-ATPase has dual functions. It not only pumps Na+ and K+ across cell membranes, but also relays the extracellular CTS signal to intracellular compartments via activation of different protein kinases.
Specifically, the inventors discovered that the Na+/K+-ATPase interacts with Src and Src family kinases to form a functional receptor. Binding of ouabain to this receptor activates Src, which in turn phosphorylates various effectors, resulting in the assembly and activation of different pathways including the Ras/Raf/ERK1/2 and phospholipase C/protein kinase C cascades as well as increases in intracellular Ca2+ and cellular ROS production. The activation of these signaling pathways eventually leads to changes in cardiac and renal functions, stimulation of cell proliferation and tissue fibrosis, protection of tissue against ischemia/reperfusion injury and inhibition of cancer cell growth. These effects occur in a tissue/cell-specific manner.
Because Src and Src family kinases play an important role in cellular signal transduction, many researchers are engaged in searching for kinase-specific and pathway-specific inhibitors. So far, many inhibitors have been developed, and most of them are developed as ATP analogs that compete for ATP binding to these kinases, resulting in inhibition of kinase activity. However, the lack of pathway specificity is a major disadvantage of the current Src inhibitors. Since Src and Src family kinases are essential for many cellular functions, a generic inhibition could compromise the overall benefit of the treatment. In the past, this has been evident by severe side effects of these inhibitors in animal studies. In addition, some of these inhibitors exhibit cross-activity toward receptor tyrosine kinases.
Cardiotonic steroids have been used as drugs to treat congestive heart failure and other cardiac diseases because they increase intracellular Ca2+ and thus contractility. However, these chemicals not only activate Na+/K+-ATPase-related cellular signaling pathways, but also inhibit the ion pumping function of Na+/K+-ATPase. The latter contributes to their clinical side effects and limits the clinical applications of these drugs. Endogenous cardiotonic steroids are hormones that regulate renal and cardiovascular functions. Over-stimulation of the newly discovered Na+/K+-ATPase/Src by these hormones is known to cause high blood pressure and induce abnormal cell proliferation in renal epithethial cells as well as induce tissue fibrosis.
Considering the above-mentioned concerns, it is clear that there remains a need in the art for a method of developing a pathway (e.g., Na+/K+-ATPase)-specific Src inhibitor or activator that can be used to block endogenous CTS-activated Src pathways or stimulate the Na+/K+-ATPase-associated Src to mimic the CTS effect without inhibiting the ion pumping function of Na+/K+-ATPase. Moreover, there is a need for targeting the newly discovered Na+/K+-ATPase/Src receptor complex to develop novel agonists or antagonists of the receptor so that the receptor function of Na+/K+-ATPase/Src complex can be either stimulated for treating diseases such as congestive heart failure and ischemia/reperfusion injury or inhibited for treating diseases such as tissue fibrosis and cancer.
There is also a need for assays to monitor Src interaction with the Na+/K+-ATPase and kinase enzymatic activities that are sensitive, simple to use, and adaptable to high-throughput screening methods.
There is also a need for a method for isolating operationally defined ligands involved in protein-protein interactions and for optimally identifying an exhaustive set of modular domain-containing proteins implicated in binding with the ligands.
If such a method were available, however, such a method would be useful for the isolation of any polypeptide having a functioning version of any functional domain of interest.
Such a general method would be of tremendous utility in that whole families of related proteins each with its own version of the functional domain of interest could be identified. Knowledge of such related proteins would contribute greatly to our understanding of various physiological processes, including cell growth or death, malignancy, renal/cardiovascular function and immune reactions, to name a few.
Such a method would also contribute to the development of increasingly more effective therapeutic, diagnostic, or prophylactic agents having fewer side effects.
According to the present invention, just such a method is provided.